Automated cell processor

ABSTRACT

Compositions and methods of treating mammalian diseases using myoblasts, and/or their physical, genetic, chemical derivatives. Myogenic cells that are normal, or genetically or phenotypically altered are cultured and transplanted into malfunctioning and/or degenerative tissues or organs to alleviate conditions that are hereditary, degenerative, debilitating, undesirable, and/or fatal. Treatment of these conditions is not limited to the usage of mechanical, electrical or physical properties of these myogenic cells, but includes the usage of biochemicals secreted/released by the latter. The present invention discloses the use of normal myoblasts to deliver the complete normal genome to effect genetic repair, or to augment the size, or the function of tissues or organs. Certain conditions may be better served with genetically altered myogenic cells derived from gene transduction, whereas others may be better served with cytoclimes converter cells. Endogenous biochemical(s) are used to control cell fusion of myoblasts among themselves or with other cell types. An automated cell processor within a cell bank which enables the manufacture, at a single run, of unprecedented large quantities (greater than 100 billion) of normal or genotypically or phenotypically altered myogenic cells is also disclosed.

REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/477,377,filed Dec. 7, 1995, now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/354,944, filed Dec. 13, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for treatingmammalian disease conditions that are debilitating, fatal, hereditary,degenerative and/or undesirable. More specifically, the presentinvention relates to the transplantation of normal, or geneticallytransduced, or cytocline-converted myogenic cells into malfunctioning,and/or degenerative tissues or organs.

2. Description of the Prior Art

MYOBLAST PROPERTIES

In mammals, myoblasts are the only cell type which divide extensively,migrate, fuse naturally to form syncytia, lose their majorhistocompatibility Class I (MHC 1) antigens soon after fusion, anddevelop to occupy 50% of the body weight in humans. These combinedproperties render myoblasts ideal for gene transfer and somatic celltherapy (SCT). Myoblast therapy is a combined SCT and gene therapy.

MYOBLAST THERAPY

Although the role of myoblasts/satellite cells in myogenesis and muscleregeneration dated back to the early 1960s (Konigsberg, I. R., Science,140:1273 (1963). Mauro, A. J., Biophys. Biochem. Cytol., 9:493-495(1961)), their use in animal therapy was not reported until 1978 (Law,P. K., Exp. Neurol., 60:231-243 1978)).

The first myoblast transfer therapy (MTT) on a Duchenne musculardystrophy (DMD) boy on Feb. 15, 1990 marked the first clinical trial onhuman gene transfer. Its success was reported (Law, P. K. et al.,Lancet, 336:114-115 (1990); Kolata, G. The New York Times, Sunday, (Jun.3, 1990)). Unlike bone marrow transplant which strictly replacesgenetically abnormal cells with normal ones, MTT actually inserts,through natural cell fusion, all the normal genes within the nuclei ofthe donor myoblasts into the dystrophic myofibers to repair them. Inaddition, donor myoblasts also fuse among themselves, forminggenetically normal myofibers to replenish degenerated ones. Thus, fullcomplements of normal genes are integrated, through a naturaldevelopmental process of regeneration, into the abnormal cells and intothe abnormal organ.

The U.S. Patent Office issued to this inventor a patent (U.S. Pat. No.5,130,141) entitled “Composition for and methods of treating muscledegeneration and weakness” on Jul. 14, 1992.

In October, 1993, the Food and Drug Administration (FDA) officiallybegan regulating somatic cell therapy (SCT) with a definition of“autologous, allogenic, or xenogeneic cells that have been propagated,expanded, selected, pharmacologically treated, or otherwise altered inbiological characteristics ex vivo to be administered to humans andapplicable to the prevention, treatment, cure, diagnosis, or mitigationof disease or injuries.” (Federal Register, 58:53248-53251 (1993)).

MTT falls under the umbrella of SCT and myoblasts and its physical,genetic or chemical derivatives become potential biologics in thetreatment of mammalian diseases.

As of May 25, 1994 the FDA has granted permission for Cell TherapyResearch Foundation (CTRF) to charge $63,806 per subject. CTRF is annon-profit 501 (c) (3) research foundation founded by the inventor in1991. Authorization by the FDA for CTRF to recover costs from subjectsof these clinical trials is extremely important to establish thescientific credibility MTT and CTRF deserve, quoting the Jun. 17, 1994edition of the Memphis Health Care News, “Permission to bill for anInvestigational product is granted rarely,” says FDA spokesman MonicaRevelle, “Applicants must endure numerous procedures, and must have whatlooks like a viable product at the end of the rainbow. It's used mainlyto support testing of promising technology by small companies.” Thisstatement was made in regard to research at CTRF.

At this time CTRF holds the first and only FDA-approved human clinicaltrial under an Investigational New Drug (IND) application on MTT. It isextremely important to realize that CTRF has been working closely withthe FDA to establish criteria and policies in the approval process ofthis IND for genetic cell therapy. The use of viral vector mediated genetherapy on human neuromuscular diseases has not met FDA approval.

CELL THERAPY WITH MYOBLASTS

The cell is the basic unit of all lives. It is that infinitely smallentity which life is made of. With the immense wisdom and knowledge ofthe human race, we have not been able to produce a living cell fromnonliving ingredients such as DNA, ions, and biochemicals.

Cell Culture is the only method known to man for the replication ofcells in vitro. With proper techniques and precautions, normal ortransformed cells can be cultured in sufficient quantity to repair, andto replenish degenerates and wounds.

Cell transplantation bridges the gap between in vitro and in vivosystems, and allows propagation of “new life” in degenerative tissues ororgans of the living yet genetically defective or injured body.

Cell fusion transfers all the normal genes within the nucleus likedelivering a repair kit to the abnormal cell. It is important torecognize that, for proper installation and future operation, thesoftware packaged in the chromosomes needs other cell organelles ashardware to operate.

Correction of a gene defect occurs spontaneously at the cellular levelafter cell fusion. The natural integration, regulation and expression ofthe full complement of over 80,000 normal genes impart the normalphenotypes onto the heterokaryon. Protein(s) or factor(s) that were notproduced by the host genome because of the genetic defect are nowproduced by the donor genome that is normal. Various cofactors derivedfrom expression of the other genes corroborate to restore the normalphenotype.

GENE THERAPY WITH MYOBLASTS

The use of myoblasts as gene transfer vehicles has been researched bythis inventor extensively. In mammals, myoblasts are the only cell typewhich divide extensively, migrate, fuse naturally to form syncytia, loseMHC-1 antigens soon after fusion, and develop to occupy 50% of the bodyweight in humans. These combined properties render myoblasts ideal forgene transfer.

Natural transduction of normal nuclei ensures orderly replacement ofdystrophin and related proteins at the cellular level in DMD. This idealgene transfer procedure is unique to muscle. After all, only myoblastscan fuse and only muscle fibers are multinucleated in the human body. Byharnessing these intrinsic properties, MTT transfers all normal genes toeffect genetic repair. Since donor myoblasts also fuse among themselvesto form normal fibers in MTT, the muscles benefit from the addition ofgenetically normal cells as well.

MYOBLAST THERAPY IS THE MEDICINE OF THE FUTURE

Health is the well-being of all body cells. In hereditary ordegenerative diseases, sick cells need repairing and dead cells needreplacing for health maintenance.

Cell culture is the only way to generate new, live cells that arecapable of surviving, developing and functioning in the body aftertransplantation, replacing degenerated cells that are lost.

Myoblasts are the only cells in the body capable of natural cell fusion.The latter allows the transfer of all of the normal genes intogenetically defective cells to effect phenotypic repair throughcomplementation. MTT on DMD is the first human gene therapy demonstratedto be safe and effective. The use of MTT to transfer any other genes andtheir promoters/enhancers to treat other forms of diseases is underway.Myoblasts are efficient, safe and universal gene transfer vehicles,being endogenous to the body. Since a foreign gene always exerts itseffect on a cell, cell therapy will always be the common pathway tohealth. After all, cels are what life is made of.

DMD: A SAMPLE DISEASE

DMD is a hereditary, degenerative, debilitating, fatal, and undesirablemammalian disease. It is characterized by progressive muscledegeneration and loss of strength. These symptoms begin at 3 years ofage or younger and continue throughout the course of the disease.Debilitating and fatal, DMD affects 1 in 3300 live male births, and isthe second most common lethal hereditary disease in humans. DMDindividuals are typically wheelchair-bound by age 12, and 75% die beforeage 20. Pneumonia usually is the immediate cause of death, withunderlying respiratory muscle degeneration and failure of DMDindividuals to inhale sufficient oxygen and to expel lung infections.Cardiomyopathic symptoms develop by mid-adolescence in about 10% of theDMD population. By age 18, all DMD individuals develop cardiomyopathy,but cardiac failure is seldom the primary cause of death.

Before 1950, over 80 chemicals were evaluated and 33 were reported aspotentially beneficial (Milhorat, A. T., Medical Annals of the Districtof Columbia, 23:15 (1954)). None are currently being used (Wood, D. S.,In: Kakulas, B. A. and Mastaglia, F. L., eds.: Pathogenesis and therapyof Duchenne and Becker muscular dystrophy. New York: Raven Press; 85-99(1990)). Unconfirmed therapeutic benefits in DMD have been reported withvitamins, amino acids (Van Meter, J. R., Calif. Med., 79:297 (1953)),ATP (Nakahara, M., Arzneim. Forsch., 15:591(1965)), coenzyme Q (Folkers,K. et al., Excerpta Med. Int. Congr. Ser., 334:158 (1974)),adenylosuccinic acid (Bonsett, C. A., Indiana Medicine, 79:236 (1986))and growth hormone inhibitor (Coakley, J. H. et al., Lancet, 1(8578):184 (1988)). Several hundred drugs have been screened (Wood, supra),with some studies showing consistent benefits from steroids (Entrikin,R. K. et al., Muscle Nerve, 7:130-136 (1984)).

The beneficial effects of prednisone on DMD was first reported almost 20years ago (Drachman, D. B. et al., Lancet, 2:1409-1412 (1974)). Theresearchers reported that prednisone could delay the degenerativeprocess and in some cases even transiently strengthen DMD muscles. Theevidence substantiating prednisone is not without debate (see Munsat, T.L. and Walton, J. N., Lancet, 1:276-277 (1985); Rowland, L. P., Lancet,1:277 (1975); and Siegel, I. M. et al., I. M. J., 145:32-33 (1974)).Although the mechanism(s) through which prednisone mediates its effectis undefined. Prednisone causes numerous side-effects, and prolonged useinduces adverse reactions that by far out-weigh the questionablebenefits reported.

Gene manipulation and transfer are other approaches that are being usedto treat hereditary and degenerative diseases. However, it will be quitesome time before this type of treatment finds clinical application forDMD (Law, P. K., In: Kakulas, B. A. et al., eds.: Pathogenesis andtherapy of Duchenne and Becker muscular dystrophy. New York: RavenPress, 190 (1990); and Watson, J. D. et al., Recombinant DNA. New York:W. H. Freeman and Co.; 576 (1992)). Success claimed over intramuscularDNA injections (Acsadi, G. et al., Nature, 352: 815-818 (1991); andWolff, J. A. et al., Science, 247:1465-1468 (1990)) and arterialdelivery of immature muscle cells, also known as myoblasts, to skeletalmuscle (Neumeyer, A. M. et al., Neurology, 42:2258-2262 (1992)) is verylimited and questionable. Attempt of using transfected autologousmyoblasts has resulted in low efficiency and mutation in transfection(Barr, E. and Leiden, J. M., Science, 254: 1507-1509 (1991); Dhawan, J.et al., Science, 254: 1509-1512 (1991); and Smith, B. F. et al., Mol.Cell. Biol., 10: 3268-3271 (1990)). Such approach will yieldinsufficient myogenic cells to provide for a whole body myoblasttransfer therapy (MTT) to treat DMD patients (Law, supra). Clinicaltrials are currently underway for cystic fibrosis (CF) based ontransgenic mice studies (Hyde, S. C. et al., Nature, 362:250-255-(1993)). Clinical trials with gene therapy have also beenattempted on severe combined immunodeficiency. (SCID). Unlike CF andSCID whose genetic defects are mediated through enzymic deficiencies,the genetic defect of DMD manifested as the absence of a structuralprotein called dystrophin in the cell membrane rather than a regulatoryprotein.

Although dystrophin serves as a good genetic/biochemical marker(Hoffman, E. P. et al., Cell, 51: 919-928 (1987)) in the evaluation ofmuscle improvements, dystrophin replacement constitutes only part of thetreatment process. This has already been demonstrated, among others, bythe present inventor using MTT in mdx mice (Chen, M. et al., CellTransplantation, 1:17-22 (1992); Karpati, G. et al., Am. J. Pathol.,135: 27-32 (1989); and Patridge, T. A., et al., Nature, 337:176-179(1989)) and in humans (Gussoni, E., et al., Nature, 356: 435-438 (1992);Huard, J. et al., Clin. Sci., 81:287-288(1991); Huard, J. et al., MuscleNerve, 15:550-560 (1992); Law, P. K. et al., Lancet, 336:114-115 (1990);Law, P. K. et al., Acta Paediatr. Jpn., 33:206-215 (1991); Law, P. K. etal., Adv. Clin. Neurosci., 2:463-470 (1992); Law, P. K. et al., In:Angelini, C. et al., eds. Muscular dystrophy research. New York:Elsevier Science Publishers, 109-116 (1991); and Law, P. K. et al., ActaCardiomiologica, 3:281-301 (1991)). Because DMD pathology is one ofmuscle degeneration and weakness, structural and especially functionalimprovements are of primary concern. These two parameters have beenextensively studied using the dy^(2J)dy^(2J) dystrophic mouse as ananimal model of hereditary muscle degeneration (Law, P. K., Exp.Neurol., 60:231-243 (1978); Law, P. K., Muscle Nerve, 5:619-627 (1982);Law, P. K. et al., Transplant Proc., 20:1114-1119 (1988); Law, P. K. etal., In: Griggs, R. C.; Karpati, G., eds. Myoblast Transfer Therapy.New: Plenum Press; 75-87 (1990); Law, P. K. et al., Muscle Nerve,11:525-533 (1988); Law, P. K. et al., In: Kakulas, B. A.; Mastaglia, F.L., eds. Pathogenesis and therapy of Duchenne and Becker musculardystrophy. New York: Raven. Press; 101-118 (1990); and Law, P. K. andYap, J. L., Muscle Nerve, 2:356-363 (1979)). These studies lead to thefirst MTT clinical trial or single muscle treatment (SMT) (Gussoni, E.et al., Nature, 356:435-438 (1992); Huard, J. et al., Clin. Sci.,81:287-288 (1991); Huard, J. et al., Muscle Nerve, 15:550-560 (1992);Law, P. K. et al., Lancet, 336:114-115 (1990); Law, P. K. et al., ActaPaediatr. Jpn., 33:206-215 (1991); Law, P. K. et al., Adv. Clin.Neurosci., 2:463-470 (1992); Law, P. K. et al., In: Angelini, C. et al.,eds. Muscular dystrophy research. New York: Elsevier Science Publishers:109-116 (1991); and Law, P. K. et al., Acta Cardiomiologica 3:281-301(1991)).

The feasibility, safety, and efficacy of MTT were assessed by thisinventor in experimental lower body treatments involving 32 DMD boysaged 6-14 years of age, half of whom were non-ambulatory (Law, P. K. etal., Cell Transplantation, 2;485-505 (1993)). Through 48 injections, 5billion (55.6×10⁶/mL) normal myoblasts were transferred into 22 majormuscles in both lower limbs in each of the subjects. Results at 9 monthsafter MTT indicated, interalia, that (1) MTT is a safe treatment; (2)MTT improves muscle function in DMD; and (3) more than 5 billionmyoblasts are necessary to strengthen both lower limbs of a DMD boybetween 6 and 14 years of age.

OTHER DISEASE CONDITIONS

Potentially every genetic disease can be benefited by MTT. Throughnatural cell fusion, donor myoblasts insert full complement of normalgenes into genetically abnormal cells to effect repair. Promoters andenhancers of the defective gene can be supplied or activated orrepressed to achieve gene transcription and translation with the releaseof hormone(s) or enzyme(s) from transplanted myogenic cells. Likewise,structural protein(s) can be produced to prevent or to alleviate diseaseconditions.

Alternatively transduced myoblasts can be used. The procedure consistsof a) obtaining a muscle biopsy from the patient, b) transfecting a“seed” amount of satellite cells with the normal gene, c) confirming themyogenicity of the transfected cells, d) proliferating the transfectedmyoblasts to an amount enough to produce beneficial effect and e)administering the myoblasts into the patient.

Retroviral vectors have been used to transfer genes into rat and dogmyoblasts in primary cultures under conditions that permit thetransfected myoblasts to differentiate into myotubes expressing thetransferred genes (Smith, B. F. et al., Mol. Cell Biol., 10:3268-3271(1990)). Furthermore, mice injected with murine myoblasts that aretransfected with human growth hormone (hGH) show significant levels ofhGH in both muscle and serum that are stable for 3 months (Dhawan, J. etal., Science, 254:1509-1512 (1991); Barr E. and J. M. Leiden, Science,254:1507-1509 (1991)).

The transduced myoblast transfer was inspired by a similar approach onadenosine deaminase (ADA) deficiency. In the latter situation, T cellsfrom the patient were transfected with functional ADA genes and returnedto the patient after expansion in the number of the transfected cellsthrough cell culture (Culver, K. W. et al., Transpl. Proc., 23:170-171(1991)).

Similar approach has already been tested in animals using geneticallytransduced myoblasts to treat hemophilia B (Yao, S. N. et al., GeneTherapy, 1:99-107 (1994)), cardiomyopathy (Marelli, D., CellTransplantation, 1:383-390 (1992); Koh, G. Y. et al., J. Clin. Inves.,92:1548-1554 (1993)), anemia (Hamamori, Y. et al., Human Gene Therapy,5:1349-1356 (1994)). Undoubtedly, there will be many hereditary diseasesto which myoblast therapy will apply.

Although differentiated, myoblasts are nonetheless embryonic cells thatare capable of de-differentiated or even converted. Thus, myoblasts haverecently been shown to be converted into osteoblasts with bonemorphogenetic protein-2 (Katagiri, T. et al., J. Cell Biol.,127:1755-1766 (1994)). This study demonstrates that cytocline-convertedmyoblasts can be administered to patients with bone/cartilagedegenerative diseases. Alternatively, it has been demonstrated thatmouse dermal fibroblasts can be converted to a myogenic lineage (Gibson,A. J. et al, J. Cell Sci., 108:207-214 (1995)).

The implicated usage of myoblast transfer therapy to treat cancer andtype II diabetes mellitus is described below.

WHY MYOBLAST THERAPY

In hereditary or degenerative diseases, gene defects cause cells todegenerate and die with time. An effective treatment must not onlyrepair degenerating cells, but replenish dead cells as well. This canbest be achieved by the transplantation of genetically normal cells, orsomatic cell therapy. The advent of molecular genetics favors singlegene manipulation which is currently being explored to treat geneticdiseases. Like pharmaceuticals, single gene manipulation cannotreplenish lost cells. Further, there is very limited evidence that theseapproaches can repair degenerating cells.

In U.S. Pat. No. 5,130,141, this inventor disclosed for the first timecompositions and methods for treating muscle degeneration and weakness.A composition comprised of genetically normal myoblasts from a donor wasinjected into one or more of the muscles of a host having a hereditaryneuromuscular disorder. Muscle structure and function were greatlyimproved with the injection, thereby preventing or reducing muscleweakness which is a primary cause of crippling and respiratory failurein hereditary muscular dystrophies. This transplantation of geneticallynormal muscle cells into the diseased muscles of patients withhereditary muscular dystrophy is known as MTT.

MTT differs significantly from the conventional single gene transferformat in several respects. In this latter gene therapy, single copiesof the down-sized dystrophin gene are transduced as viral conjugatesinto the mature dystrophic myofibers in which many proteins, bothstructural and regulatory, are lost previously. Multiple gene insertionis necessary to replace these lost proteins (FIG. 1). More geneinsertion is needed to produce the cofactors to regulate and to expressthese lost proteins in order to repair the degenerating cell.

SUMMARY OF THE INVENTION

The demonstration of preliminary feasibility, safety, and efficacy (Lawet. al., Cell Transplantation, 2:485 (1993)) of myoblast transfertherapy MTT prompted this inventor to initiate a whole body trial (WBT)injecting 25 billion myoblasts into each of 64 Duchenne musculardystrophy (DMD) boys and a boy with infantile facioscapulohumeraldystrophy (IFSH). The randomized double-blind clinical trial protocol,approved by the FDA (IND Phase II) and the Essex IRB involves two MTTprocedures separated by 3 to 9 months. Each procedure delivers 200injections or 12.5 billion myoblasts, to either 28 muscles in the upperbody (UBT) or to 36 muscles in the lower body (LBT). Injected musclesinclude those in the neck, shoulder, back, chest, abdomen, arms, hips,and legs. Subjects take oral cyclosporine for 3 months after each MTT.One IFSH and 10 DMD boys have received WBT and 20 more DMD boys havereceived UBT or LBT in the past 17 months with no adverse reaction.These preliminary results indicate that the WBT is feasible and safe.While blinding will continue until the end of the study as to which sideof the biceps brachii or quadriceps received myoblasts or placebo, fivesubjects have demonstrated immunocytochemical evidence of dystrophin inone of these muscle biopsies 3 to 9 months after MTT. The contralateralmuscle biopsies show no dystrophin. The pulmonary function (FVC) eithershows no deterioration, or has improved by 15 to 25% in over 80% of thesubjects 3 to 6 month after MTT. About 50% of the subjects reportbehavioral improvement in running, balancing, climbing stairs andplaying ball. One 14 yr-old DMD subject has stayed active without theneed of a wheelchair after MTT (Law, P. K. et al., Amer Soc NeuralTranspl Abst., p. 27 (1995); Law, P. K. et al., J. CellularBiochem.Supp, 21A:367, (1995)).

This demonstration of feasibility and safety in administering 30 billionmyoblasts into a human subject provides the pivotal evidence thatmyoblasts can be used as a biologic to treat human diseases. Thedemonstration of the dosage effectiveness further confirms the idea thatmyoblast therapy can be used to treat a whole variety of mammaliandiseases be it a hereditary, degenerative, debilitating, fatal, orundesirable disease condition.

The present invention provides compositions and methods for repairingdegenerating cells and replenishing lost cells in patients withhereditary or degenerative diseases, in particular those characterizedby muscle malfunction, degeneration and weakness. In practicing thepresent invention, any myogenic cell may be used, regardless of whetherit is of skeletal, smooth, or cardiac in origin. Transferred cell typesinclude myoblasts, myotubes and/or young muscle fibers. The myogeniccells may be primary-cultured or cloned from muscle biopsies of normaldonors. They may also be cytocline converted or genetically transducedmyogenic cells. Typically, the parents, siblings, or friends of thedystrophic patient are the donors. In addition, it is contemplated thatthe establishment of superior cell. lines of myoblasts, whether fromhumans or animals, will provide a ready access of healthy donor cellsfor patients who do not otherwise have a suitable donor (FIGS. 2 to 5,also Law, P. K., Myoblast Transfer, Landes, Austin, (1994)). It isfurther contemplated that the cell transplantation procedure willaugment size, shape, appearance or function, and/or alleviate thedisease conditions.

The present invention provides a method for controlling, initiating, orfacilitating cell fusion once the myoblasts are injected into one ormore of the muscles of a patient with the degenerative disease. Byartificially increasing the concentration of the largechondroitin-6-sulfate proteoglycan (LC6SP) over the patient's endogenouslevel, fusion of the transferred donor myoblasts among themselves orwith other cell types can be enhanced and controlled. (Law, P. K.,Myoblast Transfer Landes, Austin (1994)). It is yet another object ofthe invention to improve the fusion rate between the host and donorcells. To this end, various injection methods were tested and comparedincluding injecting diagonally through the myofibers, perpendicular tothe myofiber surface, parallel to the myofibers, and at a single siteinto the muscle. The goal is to achieve maximum cell fusion with theleast number of injections (FIGS. 6 to 8, also Law, P. K., MyoblastTransfer, Landes Austin, (1994)).

In a further embodiment, the technologies of in vitro fertilization andblastomere recombination can be used on known Duchenne carriers toincrgease their chances of having normal children (FIGS. 9 to 13; alsoLaw, P. K., Myoblast Transfer, Landes, Austin, (1994)).

It is yet another object of the present invention to provide anautomated cell processor, a highly efficient means for producing massquantities (over 100 billion in one run) of viable, sterile, geneticallynormal as well as functional myogenic cells whether geneticallytransduced or cytocline-converted. The cell processor has an intakesystem which will hold biopsies of various human tissues. The cellprocessor's computer will be programmed to process tissue(s), and willcontrol time, space, proportions of culture constituents and apparatusfunctions. Cell conditions can be monitored at any time during theprocess. The output system provides a supply of cells suitable fortransfer in cell therapy or for shipment (FIGS. 14 to 15).

It is yet another embodiment in which myoblasts, and/or their physical,genetic, chemical derivatives, are used to treat cancer. FIGS. 16 to 18illustrate melanoma cancer cell death upon exposure to myoblasts infusion medium. According to Cancer Prevention and Control edited byGreenwald, P., Kramer, B. S., and Weed, D. L. (Marcel Dekker, Inc. NewYork, 1995), skeletal muscles appear to be devoid of cancer, thoughmalignant tumor and metastases are found in every other organ. The veryfew cases of sarcoma reported are rare exceptions.

The recent immunocytochemical demonstration of dystrophin in DMD muscles9 months after MTT indicated long term correction of genetic defect(s)can result from myoblast therapy (FIG. 19). This principle can apply totreat malfunctional insulin resistant muscles in Type II diabetesmellitus. As a universal gene transfer vehicle, donor myoblasts insertthe whole normal genome and this can repair any malfunction of skeletalmuscle cells, rendering them insulin sensitive (FIG. 20).

Additional features and advantages are described in and will be apparentfrom the detailed description of the presently preferred embodiments andfrom the drawings. Further, all references described herein areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of some of the known protein defects in DMD musclecells that differ from normal muscle cells. These include membranestructural proteins that are decreased or absent in DMD such asdystrophin (DIN), dystrophin-related-protein (DRP) anddystrophin-associated-glycoproteins (DAG), enzymes elevated in serumlevels of DMD patients such as creatinine phosphokinase (CPK), aldolase(AID) and aspartate transaminase (AST), and mitochondrial (Mito) proteindifferences. Although these protein defects are caused primarily orsecondarily by the dystrophin gene defect, their correction will requiremultiple gene (G1 to G7) transfer.

FIG. 2 illustrates MHC-negative myoblasts and MHC-positive myoblasts.(A) represents human myoblasts assayed with anti-MHC class I antibodiesand viewed under fluorescent microscopy. The arrow indicates theMHC-negative myoblast. (B) is of the same slide viewed under a regularlight microscope. The arrow indicates the MHC-negative myoblast. Bar=20μm.

FIG. 3 illustrates an analysis of myoblasts by cytofluorometry. Control:myoblasts reacted without anti-MHC class I antibodies. Samples:myoblasts reacted with anti-MHC class I antibodies.

FIG. 4 is a scatter diagram of the separation of the myoblasts that werenegative for MHC-I antigen expression by cytofluorometry.

FIG. 5 illustrates fluorescent intensities of two groups of myoblastsafter separation by cytofluorometry. (A) represents MHC-positivemyoblasts. (B) represents MHC-negative or weakly expressed myoblasts.(A) and (B) are of the same magnification and the pictures weresimilarly processed. Bar=30 μm.

FIG. 6 illustrates the angle of injection that determines myoblastdistribution. (A) Oblique myoblast injection delivers donor myoblasts tothe greatest number and area of recipient muscle fibers with the leastleakage, resulting in the formation of the most mosaic fibers. (B)Transverse injection delivers donor myoblasts to a large number offibers, but covers a smaller area and is more likely to result inleakage from the injection. (C) Longitudinal injection results in donormyoblasts fusing with each other, with fewer mosaic fibers being formed.(D) Focal injection results in only a small area of a few recipientmuscle fibers being injected with donor myoblasts.

FIG. 7 illustrates donor myoblast nuclei labeled with fluoro-gold (FG),and are present in host muscle at seven days after MTT. (A) Mosaicmyofiber with donor and recipient nuclei (black arrow) and donor myotubewith, donor myonuclei (white arrow). (B) Mosaic myofibers with donor(white arrows) and recipient (black arrows) nuclei.

FIG. 8 illustrates distributions of donor myoblasts labeled with FG inhost muscle. Even distribution of donor myoblasts can be achieved withoblique myoblast injection (A,B), or donor nuclei may appear in patches(C,D, white arrows) which gradually show more abnormal nuclei anddebris. (C) represents a transverse injection and (D) a longitudinalinjection.

FIG. 9 illustrates the production of allophenic twins with the mechanismof allophene formation.

FIG. 10 illustrates three littermates: 1 normal, 1 allophene, 1dystrophic (top to bottom).

FIG. 11 illustrates physiological recordings of maximal isometric twitchand tetanus tensions at 80 Hz elicited from the soleus muscles of thenormal, allophenic, and dystrophic littermates. The allophene recordingsresemble the normal, rather than the dystrophic recordings.

FIG. 12 illustrates soleus cross-sections from allophenic micedemonstrating core fibers (arrows) characteristic of muscle from DMDcarriers. Whereas most myofibers remain normal in appearance, somedegenerative characteristics such as fiber splitting and centralnucleation are apparent.

FIG. 13 further illustrates soleus cross-sections from allophenic micedemonstrating core fibers (arrows) characteristic of muscle from DMDcarriers. Although most myofibers remain normal in appearance, somedegenerative characteristics are visible such as fiber splitting andcentral nucleation.

FIG. 14 illustrates the general layout of an automated cell processor.

FIG. 15 illustrates the detailed design of culture and harvest automatedactions.

FIG. 16 is a comparison of cancer cell growth media with myoblast growthmedia on cultured melanoma (CRL6322) cells. (A, C, E, G) lowmagnification; (B, D, F, H) high magnification. (A, B) Melanoma cellscultured in cancer cell growth media for 9 days appear healthy, asevidenced by their numbers, elongated shapes, and branching processes.(C, D) Similar amount of melanoma cells seeded and cultured in myoblastgrowth media for 9 days are more numerous and differentiated. (E, F)After 14 days in cancer cell growth media, the melanoma cells, whilenumerous, have become spherical in shape and detached from the surface,indicating that the cells are dead (E). At higher magnification only afew healthy cells remain (F). (G, H) After 14 days in myoblast growthmedia, the melanoma cells still appear numerous and healthy.

FIG. 17 illustrates myoblasts and melanoma cells (2:1 concentrationratio) co-cultured in myoblast growth media. (A, C) low magnification;(B, D) high magnification. (A, B) After 9 days in culture myoblasts andmelanoma cells are numerous and remain differentiated. (C, D) After 14days in culture myoblasts dominate the culture, with melanoma cellsstill surviving.

FIG. 18 illustrates myoblasts and melanoma cells co-cultured in myoblastgrowth medium for 4 days and then in myoblast fusion medium. (A, C, E,G) low magnification; (B, D, F, H) high magnification. (A, B) Myoblastsand melanoma cells (1:1 concentration ratio) after 5 days in myoblastfusion medium. Many melanoma cells have become spherical and detachedfrom the surface and are not surviving in this medium. Myoblasts remainhealthy and are beginning to fuse. (C, D) Myoblasts and melanoma cells(1:1 concentration ratio) after 10 days in myoblast fusion medium shownumerous dying cells. Only a few can survive in this medium for thislong, and they are detached and dying (D). (E, F) Myoblasts and melanomacells (1:1 concentration ratio) after 19 days in myoblast fusion media.The surviving myobalasts retain their spindle shape and align with eachother. Melanoma cells have become spherical and detached. (G, H)Myoblasts and melanoma cells (3:1 concentration ratio) after 19 days inmyoblast fusion medium. Myoblasts have begun to fuse, forming myotubes.

FIG. 19 is an immunocytochemical demonstration of dystrophin in humanmuscle. Sarcolemmal localization of dystrophin is shown in normalcontrol muscle (A) but not in DMD muscle (B). (C, D) DMD biceps brachiimuscles from a subject who received MTT in one biceps and placeboinjections in the contralateral muscle 9 mo before biopsies. Sinceblinding continues until the end of the study, the designation of theMTT muscle cannot be revealed. However, only one muscle showssarcolemmal localization of dystrophin. (B), (C), and (D) wereintentionally over-exposed to show immuno-reactive background elementsnot associated with the sarcolemma.

FIG. 20 illustrates (a) normal skeletal muscle metabolizing glucose withinsulin. (b) In Type II diabetes mellitus, the major sequela of insulinresistance is decreased muscle uptake of glucose. It is possible thatthe glucose transporter in muscle is abnormal. It is known thatinsulin-mediated stimulation of tyrosine kinase and autophosporylationare impaired. These latter defects can. be corrected by MTT by normalgene expression (from Metabolism 6:6, 1993).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compositions and methods described herein will be illustrated forthe treatment of individuals having hereditary neuromuscular diseases.However, it is contemplated that other hosts and other disease statesmay be treated with the inventive compositions and methods.

A. CONTROLLED CELL FUSION

Myoblasts have the unique ability to fuse with other cells. With the useof normal myoblasts, a full complement of normal genes can be introducedinto any genetically abnormal cells through cell fusion. For example,the genetically abnormal cell could be a liver cell, heart muscle cell,or even a brain cell. The idea is to introduce a full complement ofnormal genes into abnormal cells and, therefore, treat the geneticdisease at the gene level and not at the hormonal or biochemical level.

For treating genetic diseases that involve structural proteinabnormalities rather than regulatory protein abnormalities, it would beuseful to control, initiate or facilitate cell fusion once myoblastswere injected into the body. It is known that myoblasts fuse readily atlow serum concentrations in culture. The process is more complex in thein vivo situation. As the myoblasts are injected intramuscularly intothe extracellular matrix (ECM), injection trauma causes the release ofbasic fibroblast growth factor (bFGF) and large chondroitin-6-sulfateproteoglycan (LC6SP) (Young, H. E., et al., J. Morph., 201:85-103(1989)). These latter growth factors stimulate myoblasts proliferation.Unfortunately, they also stimulate the proliferation of fibroblasts thatare already present in increased amounts in the dystrophic muscle. Itis, therefore, necessary to inject as pure as possible fractions ofmyoblasts in MTT without contaminating fibroblasts.

Controlled cell fusion can be achieved by artificially increasing thelocal concentration of LC6SP over the endogenous level at the transfersite. In muscles, this is achieved by including approximately 5 μM ofLC6SP in the transfer medium. In addition, insulin facilitates thedevelopmental process in vitro, and may result in the formation ofmyotubes soon after myoblast injections. The use of LC6SP (ranging fromapproximately 5 μM to about 5 mM) in the transfer medium will likelylead to greater MTT success.

B. MYOBLASTS: THE UNIVERSAL GENE TRANSFER VEHICLES

Whereas MTT results in the formation of genetic mosaicism with genetransfer occurring in vivo, the production of heterokaryons in vitro hasimmense medical application. This can be achieved by controlled cellfusion with myoblasts.

This research relates to the in culturo transfer of the normal nucleiwith all of their normal genes from donor myoblasts into the geneticallynormal and/or abnormal cells, e.g. the cardiomyocytes. This developmentis especially important considering that cardiomyopathic symptomsdevelop in mid- adolescence in about 10% of the DMD population. By age18, all DMD individuals develop cardiomyopathy. Undoubtedly, the abilityto replenish degenerated and degenerating cardiomyocytes will have animmense impact on heart diseases even in the normal population wherethere is a great shortage of hearts for transplantation.

Normal cardiomyocytes have a very limited ability to proliferate in vivoor in vitro. The heart muscles damaged in heart attacks or in hereditarycardiomyopathy cannot repair themselves through regeneration. Byintegrating the skeletal muscle cell characteristic, mitosis,heterkaryotic cardiomyocytes will be able to proliferate in vitro.

Controlled cell fusion between normal myoblasts and normalcardiomyocytes may result in heterokaryons exhibiting thecharacteristics of both parental myogenic cell types. Clones can beselected based on their abilities to undergo mitosis in vitro to developdesmosomes, gap junctions, and to contract strongly in synchrony aftercell transplantation.

These genetically superior cells can then be delivered through catheterpathways of the type described by Jackman WM, et al. (In: Zipes DP, andJalife J, eds. Cardiac electrophysiology. From Cell to Bedside.Philadelphia: WB Saunders Company, 491-502, (1990)) after mapping of theinjured sites. With the ability to grow large quantity of thesecardiomyocytes, the correction of structural, electrical and.contractile abnormalities in cardiomyopathy can be tested first indystrophic, cardiomyopathic hamsters and then in humans.

The genetic transfer of the mitotic property of myoblasts ontocardiomyocytes with in vitro controlled cell fusion enables theresulting heterokaryotic cardiomyocytes to multiply, yielding enoughnumbers of cells for the cell transplant to be effective.

Recently, it was reported that fetal mouse cardiomyocytes grafted intothe myocardium of syngeneic hosts formed nascent intercalated disksbetween host and donor cells (Soonpaa MH, et al., Science,264:98-(1994)). The use of fetal cells for cell transplant has and willcontinue to raise ethical questions. The fact still remains that fetalcells will not produce enough cardiomyocytes to mend a myocardialinfarct. The bioengineering of mitotic cardiomyocytes using myoblastsprovides a solution to the problem in view of reported studies thatrecombinant genes introduced into cardiomyocytes are expressed for atleast 6 months, and appear to be regulated normally by humoral signals.

Whereas myoblast transfer into the dystrophic myocardium followed by invivo controlled cell fusion may provide a structural impediment at theinfarct, it remains to be shown that the myoblasts will integrate wellwith the cardiomyocytes, considering that the pumping action of theheart will disaggregate the developing cells from the host myocardium.

C. COSMETIC USAGE

In a broader sense, the cell therapy concept can significantlycontribute to the field of plastic surgery. With cell therapy,implantation of silicone could be avoided. The use of myoblasts and/orfat cells could be used in a much more natural way to replace siliconeinjections for facial, breast and hip augmentation. Modified adiposetissue involving mixing and/or hybridization of myoblasts and fat cellscan be used to control size, shape and consistency of body parts. Sincemuscle cells do not break down as easily as fat cells, good results maybe long-lasting. Today, body builders are in search of increasing musclemass and function at the right places. The use of myoblast transfer toboost muscle mass is a natural solution.

D. SUPERIOR CELL LINES

The establishment of superior cell lines of myoblasts is a high-riskchallenge, but its benefits are numerous. These cell lines should behighly myogenic, nontumorigenic, nonantigenic, and will develop verystrong muscles.

A unique property of myoblasts is their loss of major histocompatibilitycomplex class I (MHC-I) surface antigens soon after they fuse. This hasimportant implications in the usage of an immunosuppressant aftermyoblast transfer therapy. (See Huard J, et al., Muscle Nerve, 17:224-34(1994); Roy R, et al., Transpl Proc, 25:995-7 (1993); and Huard J, etal. Transpl Proc, 24:3049-51 (1992)). The immunosuppression perioddepends on how soon the myoblasts lose their MHC-I antigens after MTT.Even more ideal is the establishment of a myoblast cell line in whichMHC-I antigens are absent, thereby allowing MTT withoutimmunosuppression.

In our study, human myoblasts were cultured from normal muscle biopsiesin accordance with the methods disclosed in Law, P. K., et al., CellTransplantation, 1:235 (1992) and Law, P. K., et al., CellTransplantation, 2:485 (1993). The MHC-I antigens expressed on themyoblasts were demonstrated with fluorescent immunoassay. Cell cyclesynchronization of myoblasts was carried out by adding colchicin in thegrowth medium and incubating for 48 hours. The myoblast preparationsused in the experiment were 98% pure as assessed by immunostaining withthe monoclonal antibody (MAb) anti-Leu-19.

Myoblasts were incubated with anti-MHC-I MAb (mouse 1:25 dilution,Silenus Lab, Australia) at room temperature. After washing, themyoblasts were incubated with FITC conjugated anti-mouse-IgG (Sigma) for45 minutes and examined under fluorescence microscope with wide bandultraviolet (UV) excitation filter. Cytofluorometry was performed with aBecton-Dickinson cell sorter operated at 488 mM. Myoblast control wascarried out by omitting the first antibodies in the immunoassay as thebackground of autofluorescence.

91.7% of the myoblasts reacted with MHC-I MAb. The reactions ranged fromstrong to weak. The remaining 8.3% of the myoblasts were negative forMHC-I antigen expression. FIG. 2 illustrates both MHC-negative myoblastsand MHC-positive myoblasts. The MHC-negative myoblasts were successfullyseparated by cytofluorometry, which is illustrated in FIGS. 3,4. Bothgroups of myoblasts were then cultured for three weeks withoutsignificant difference in proliferation.

The lack of MHC-I antigens on these myoblasts may enhance survival ofthese myoblasts in recipients after MTT. FIG. 5 illustrates thefluorescent intensities of both MHC-positive myoblasts and MHC-negativeor weakly expressed myoblasts after separation by cytofluorometry.

The immunosuppressant, cyclosporine, has many side effects and bysuppressing the immune system, allows infection to prevail. Myoblastswithout MHC-I antigen expression may contribute to a new cell line morecapable of surviving in the host than the regular myoblasts. Thissuperior cell line will eliminate the need to use the immunosuppressant,and will provide a ready access for patients who do not have a donor.

These superior cell lines have to be derived from clones of primarymyoblast cultures because they are selected for their unique properties.Unfortunately, it has been shown that all clones of myoblasts eventuallyproduce tumors if allowed to proliferate excessively. Thus, these celllines should not be allowed to proliferate over 30 generations.

E. MYOBLAST INJECTION METHODS

Aside from donor cell survival in an immunologically hostile host, cellfusion is the key to strengthening dystrophic muscles with MTT. Toimprove the fusion rate between host and donor cells, various injectionmethods aimed at wide dissemination of donor myoblasts were tested andcompared. These included injecting diagonally through the myofibers,perpendicular to the myofiber surface, parallel to the myofibers, and ata single site into the muscle. FIG. 6 illustrates myoblast distributionas a function of the angle of the injection. The goal was to achievemaximum cell fusion with the least number of injections.

Fluoro-gold (FG, 0.01%) labeled human or mouse (C57BL/6J-gpi-lc/c)myoblasts (0.05 ml of a 10⁵ cells/ml solution) were injected into thegastrocnemius muscles of twenty normal 3-month old normal mice(C57BL/6J-gpi-lb/b). Host mice were immunosuppressed with a dailysubcutaneous injection of cyclosporine at 50 mg/kg body weight. Groupsof mice were sacrificed on day 7, 14, 24, 34, and 44 after cellinjection. Transverse. sections of injected muscles were examined withfluorescence microscopy. The cell fusion rates were estimated bycalculating the percentage of host muscle fibers bearing donor nucleiout of the total number of muscle fibers in the area of donor cellcovered. The glucose phosphate isomerases (GPI) of the injected muscleswere also examined with agarose gel electrophoresis (200 V anode tocathode, 3 hours, pH 8.6). The first appearance of mosaic myofibers inthe tissue sections was within seven days after cell injection. This isillustrated in FIG. 7. The highest fusion rate achieved was 72.2%. Theelectrophoreograms of GPI showed host donor and mosaic GPI in musclespecimens at least up to 44 days after MTT. Myoblasts injected obliquelythrough the myofibers were widely and evenly distributed with ejectionof the myoblasts as the needle is withdrawn. This is shown in FIG. 8.Myoblasts injected perpendicular to the myofibers were partiallydistributed, while myoblasts that were injected longitudinally throughthe core of the muscles and parallel to the myofibers were poorlydistributed. Similarly, injection at one spot gave poor distribution andfusion. Considering that a small volume of a concentrated solutioncauses less muscle damage than a larger volume of a relatively lessconcentrated solution, and in view of the trauma caused by injectiondecreases the regenerative capability of dystrophic muscles, thetechnique of myoblast delivery is essential for MTT success.

Although oblique injection has been used in our clinical trials, thereis room for improvement since human muscles are larger and the myofiberorientation of different muscle groups have to be well-studied by theorthopedic surgeons who administer myoblast injections. Judging fromprevious mouse studies, 20% normal myonuclei were able to maintainnormal phenotype in dystrophic myofibers.

F. EXERCISE AND PHYSICAL THERAPY

Strenuous exercise causes damage to dystrophic myofibers. Lack ofdystrophin causes the vulnerable sarcolemma to tear upon contraction.Other cell types are somewhat spared from degeneration because they donot contract. Thus, body building is counter-productive in DMD patientsto compensate for loss of muscle mass and strength.

The use of exercise, however, in relation to MTT has not been studied.In dystrophic animals, it is well known that exercise hastens thedegeneration of myofibers and thus aggravates the dystrophic condition,that is with dystrophic muscle fibers alone. The situation is differentfrom MTT in which an attempt is made to produce a mosaic musclecontaining normal, mosaic, and dystrophic fibers. The essence of MTT isto reconstruct the genetics and improve the phenotypes of dystrophicmuscles. Thus, intensive exercise may induce the release of hostsatellite cells that will fuse with normal myoblasts to produce mosaicfibers. Undoubtedly, such dystrophic degeneration will induce normalmuscle regeneration. Implanted myoblasts not only fuse to the newlysealed regions of damaged myofibers, but also survive as satellitecells. Mild exercise done shortly after MTT can be designed tofacilitate myoblast mixing, alignment, and fusion, and to providephysical therapy to the newly formed fibers. Moderate exercise afterinnervation of newly formed fibers is likely to enhance the developmentof normal and mosaic fibers. Disuse plays a major role in the continueddeterioration of dystrophic muscles, and physical therapy is prescribedfor dystrophic patients. Disuse or lack of cross-bridge interactionresults in a decrease of calcium binding. As a result, the excessiveintracellular calcium promotes muscle damage in dystrophic muscles.

G. MYOTUBE TRANSFER

In the later stages of DMD, there remains fewer myofibers to be repairedwith MTT. Formation of new fibers to replenish degenerated cells isfurther complicated by the presence of excessive connective and fattissues. While it takes approximately 1 to 3 weeks for donor myonucleito be incorporated into dystrophic fibers for repair, it takes over 4months for donor myoblasts to develop into mature normal fibers de novoto replenish lost cells. Meanwhile, the impediment to developingmyotubes to be vascularized, innervated, and connected to tendons allthreaten their survival. Enough nutrients have to be present for thedeveloping fibers to lay down the contractile filaments myosin andactin. Neither electrical nor contractile activity is normal for thedevelopment of the fibers. This is the time when myotube transfer may beof help.

Transplants of newborn normal muscles or myotubes into dy^(2J)dy^(2J)dystrophic mouse muscles have been shown by this inventor to producenormal muscle function and structure. (See Law, P. K. and Yap, J. L.,Muscle Nerve, 2:356-63 (1979)). Myotubes are easily obtained in culturothrough natural myoblast fusion by exposing confluent cultures to thefusion medium. In fact, small muscles have been produced withspontaneously contracting fibers in culture. The young fibers exhibitsarcomeres and immunostain positively for myosin.

Myotube transfer can be administered through injection with larger gaugeneedles. Better still, they can be surgically implanted into the beds offat and connective tissues dissected and removed by surgeons. Sincemuscles can develop great forces and scar tissues are inert, thedeveloping muscles will force the scar tissues aside throughout theirexistence.

Myotube transfer provides bioengineered young fibers in vitro. Thesefibers have lost their MHC-I surface antigens and are thus nonantigenic.Myotube transfer will not need to be administered with cyclosporin. Forpatients previously infected with cytomegalovirus (CMV). or otherviruses, myotube transfer will be the choice.

In addition, for autosomal dominant diseases such as facioscapulohumeraldystrophy (FSH), myotonia congenita, myotonia dystrophica and certainforms of congenital muscular dystrophy and limb-girdle dystrophy,formation of mosaic fibers may not be useful since nuclearcomplementation may not be effective. The use of entirely normalmyotubes through myotube transfer will undoubtedly open new avenues fortreatment.

H. ALLOPHENIC MICE

Allophenic mice or mouse chimaeras are mice mosaics with two or moregenotypes. They are produced by blastomere recombination (see Hogan B.,et al. Manipulating the Mouse Embryo. A Laboratory Manual. Cold SpringHarbor Laboratory, (1986)) or by the artificial aggregation of embryosfrom two different strains of mice. In addition to being importantspecimens to study the clonal origins of somites and their musclederivatives, allophenic mice have been shown by this inventor and othersto demonstrate dystrophy suppression on natural development whengenetically normal and dystrophic myogenesis coexist.

By aggregating half embryos of normal (129 strain) and dystrophic(C57BL/6J dy^(2J)dy^(2J) strain) mice as shown in the diagram of FIG. 9,sets of allophenic twins were produced consisting of chimaeric mice andtheir normal and dystrophic littermates (FIG. 10). Although thedystrophic gene was present in the muscle fibers according to genotypemarker analyses, these allophenic mice showed normal behavior, lifespan, and essentially normal muscle function shown in FIG. 11 andstructure in FIGS. 12 and 13.

Muscle fibers of these allophenic mice highly resemble those of theDuchenne female carriers. Whereas the dystrophic soleus contains 70% ormore degenerating fibers, only 3 to 5% of the allophenic soleus fibersare abnormal. Many of these abnormal fibers showed “cores” as seen inthe Duchenne carriers. Through natural cell fusion, normal myoblastsfuse with dystrophic ones to form mosaic myotubes that develop intophenotypically normal fibers.

I. NORMAL CHILD FOR A DUCHENNE CARRIER

Conceivably the technology of in vitro fertilization and blastomererecombination used in the allophenic mouse studies can be applied tohuman. Known carriers may thus have better chances of bearing normalchildren.

Accordingly, ova from a carrier and from a normal female can be obtainedand fertilized in vitro with sperm recovered from the carrier's husband.The fertilized egg of the carrier has a 50/50 chance of being normal ordystrophic. Regardless of its genotype, its mixing with the normalfertilized egg will ensure the development of a normal phenotype. Afterculturing the embryo into the blastocyst stage, it can be implanted intothe uterus of the carrier. The latter can be induced to bepseudo-pregnant with human gonadotrophin, thus allowing easyimplantation.

The use of in vitro fertilization protects the mother. Abnormaldeveloping embryos after blastomere recombination can be discarded.Furthermore, no blastomere needs to be removed for genetic analysis.

Since the fertilized egg of the carrier has 50% chance of being normal,a PCR analysis for dystrophin messenger RNA can be conducted onblastomeres removed from the embryo at the blastocyst stage.Unfortunately, this risks damaging the embryo by removing part of it atan early developmental stage.

J. AUTOMATED CELL PROCESSORS

With the great demand for normal myoblasts, myotubes and young muscles,the labor intensiveness and high cost of cell culturing, harvesting andpackaging, and the fallibility of human imprecision, an automated cellprocessor is needed. Such a processor would be capable. of producingmass quantities, over 100 billion per run, of viable, sterile,genetically well-defined and functionally demonstrated biologics, forexample, myogenic cells.

The automated cell processor will be one of the. most importantoffspring of modern day computer science, mechanical engineering andcytogenetics (FIG. 14). The intakes will be for the biopsies of varioushuman tissues. The computer will be programmed to process tissue(s),with precision control in time, space, and proportions of cultureingredients and apparatus maneuvers. Cell conditions can be monitored atany time during the process, and flexibility is built-in to allowchanges. Different protocols can be programmed into the software forculturing, controlled cell fusion, harvesting and packaging. The outputswill supply cells, which will be ready for shipment or for injectionusing cell therapy. The automated cell processor will be self-containedin a sterile enclosure large enough to house the hardware in which cellsare cultured and manipulated (FIG. 15).

This inventor has developed a transfer medium that can sustain thesurvival and myogenicity of packaged myoblasts for up to 3 days at roomtemperature. Survival up to 7 days can be achieved when the myoblastsare refrigerated. This will allow the cell packages to be delivered toremote points of utilization around the world.

The automated cell processor will simply replace current bulkyinefficient culture equipment and elaborate manpower. Its contributionto human healthcare will undoubtedly be significant, and themanufacturing costs are expected to be relatively low.

K. CELL BANKS

The automated cell processors will constitute only a part of cell banks.Ideally, donor muscle biopsies can be obtained from young adults aged 8to 22 to feed the inputs of the automated cell processor. This willdepend on the availability of healthy volunteer donors. Each donor hasto undergo a battery of tests that are time-consuming and expensive.Based on the test results and the donor's physical condition, one candetermine if the donor cells are genetically defective or infected withviruses and/or bacteria. These are the advantages of biopsies of maturetissues from adults. The major disadvantage, however, is that maturecells often do not divide, and even if they do, there is a limitednumber of generations that can propagate before becoming tumorigenic ornonmitotic.

Human fetal tissues can potentially provide unlimited supplies ofdividing cells. However, aside from ethical issues, it is difficult todetermine the genetic normality of these cells, notwithstanding theexistence of polymerase chain reaction (PCR) which is used to screenmany human genetic diseases.

As for the muscular dystrophies, the use of muscle primordia of fetalcalves derived from in vitro fertilization of genetically well-definedbackground may be an alternative. Sperms and ova can be recovered frominbred strains of cattles that are known for their muscle strength andmass. In vitro fertilization will be followed by embryo culture andimplantation into the uteruses of pseudo-pregnant cows. The fetuses areremoved by Sicilian sections at specific developmental stages of theembryos. The muscle primordia that are rich in myoblasts can then bedissected out to feed into the automated cell processors.

Transplantation of cattle cells into humans constitutes xenografting.Due to the significant differences between the human and the cattleimmune systems, these xenografts will likely survive, develop andfunction in the recipients without the need for immunosuppressants.However, the method will be tested. with and without immunosuppressants.

L. MYOBLAST DERIVATIVES VS. CANCER

Evolution is one continual experiment through ages with numerousstatistics. The near absence of cancer metastases in skeletal musclessuggests that the physical, electrical, mechanical or chemical presenceof myogenic cells and derivatives prevents or annihilates cancer.

In our study we showed that the physical and biochemical conditions ofmyoblast at the cell fusion stage caused the death of melanoma cancercells (FIGS. 16 to 18). This does not preclude the potential effect ofsimilar or different conditions, including electrical and mechanical, ofother myogenic cells at different developmental stages.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its attendant advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

I claim:
 1. An automated cell processor for producing mammalian cellscomprising: (a) a sterile enclosure having controlled gas, temperatureand humidity and (b) multiple trays stacked parallel to each otherwithin the enclosure, each tray comprising multiple culture plates thatcontain a water solution and each plate in fluid contact with adjacentplates by one or more flow ducts, wherein a fluid is automaticallydispensed into the trays and waste fluid is removed from the trays bytilting the trays.
 2. An automated cell processor as described in claim1, wherein said trays are comprised of metal.
 3. An automated cellprocessor as described in claim 2, wherein said metal is stainlesssteel.
 4. An automated cell processor as described in claim 1, whereinsaid fluid enters one end of each tray and said waste fluid exits theopposite end of each tray.
 5. An automated cell processor as describedin claim 1, wherein one or more of said flow ducts levels said watersolution between adjacent culture plates.
 6. An automated cell processorfor producing mammalian cells comprising: (a) a sterile enclosure havingcontrolled gas, temperature and humidity; (b) multiple trays stackedparallel to each other within the enclosure, each tray comprisingmultiple culture plates and each plate in fluid contact with adjacentplates by one or more flow ducts; (c) at least one water solution thatis added to the trays within the enclosure; (d) a collection containerwithin the enclosure that receives a cell mixture from the multipletrays for harvesting; and (e) an automation controller that monitorstime, media composition and gases and that controls movement of thetrays, wherein the automation controller fills the multiple trays withthe water solution, maintains the pH, tilts the multiple trays, andtransfers cells to the collection container.
 7. An automated cellprocessor as described in claim 6, wherein said trays are comprised ofmetal.
 8. An automated cell processor as described in claim 7, whereinsaid metal is stainless steel.
 9. An automated cell processor asdescribed in claim 6, wherein said water solution enters one end of eachtray and waste fluid exits the opposite end of each tray.
 10. Anautomated cell processor as described in claim 6, wherein each flow ductlevels the solution between adjacent culture plates.
 11. An automatedcell processor as described in claim 6, wherein a surface of each trayis sterilized.
 12. An automated cell processor as described in claim 6,wherein said mammalian cells are myoblast cells.
 13. An automated cellprocessor as described in claim 6, wherein said water solution isdispensed to the trays by jets.